P. aeruginosa is one of the principal pathogens associated with Cystic fibrosis (CF) pulmonary infection and is responsible for a decline in health and poor prognosis for these patients. Once established, growth of P. aeruginosa in biofilms makes it very difficult to eradicate the organisms by antimicrobial treatment. Recent findings indicate that the progression of biofilm development coincides with marked changes in the Ser/Thr phosphorylation patterns and that the biofilm developmental process is controlled by a series of phosphorylated regulatory proteins. Furthermore, our data indicate that inactivation of biofilm-specific regulatory proteins impaired or arrested the biofilm developmental process. The goal of the proposed studies is to identify phosphorylated proteins involved in the regulation of P. aeruginosa biofilm formation that once inactivated, impair or arrest the progression of biofilm development in vitro. We hypothesize that P. aeruginosa biofilm development is controlled by a series of phosphorylated regulatory proteins and that inactivation of these regulatory proteins will not only impair the progression of biofilm development but also result in biofilms that are more susceptible to antimicrobial agents than mature biofilms. We will first identify phosphorylated regulatory proteins that are unique to the transition from planktonic growth stage to maturation-1 and -2 biofilm developmental stages. This will be achieved by purifying phosphorylated regulatory proteins from planktonic and biofilm growth stages by metaloxide affinity chromatography (MOAC), followed by 2D/PAGE and subsequent identification by mass spectrometry using peptide mass fingerprinting. To elucidate the regulatory process of biofilm formation, we will make use of insertional mutation of regulatory proteins and examine whether inactivation of these regulators impairs initial attachment and/or biofilm development under flowing conditions and how inactivation of regulatory proteins affects the protein phosphorylation and protein production patterns. Furthermore, mutant strains impaired in the progression of biofilm developmental will be tested for susceptibility to antimicrobial agents. Mutants that are more susceptible to antimicrobial agents will be further analyzed by RT-PCR to gain further insight into the mechanism of antimicrobial susceptibility by determining the transcript abundance of mexF, mexC and the transcriptional regulator PA4878. Findings from this detailed investigation of the regulation of the P. aeruginosa biofilm developmental process are expected to lead to innovative and more effective treatment strategies based on inhibition or regulation of biofilm formation to treat and control biofilm infections. Biofilms are considered the root of many persistent and chronic bacterial infections, which are both refractory to antibiotic therapy and barely affected by host defenses. Findings from this research are intended to lead to novel and more effective approaches for the treatment of such biofilm infections. [unreadable] [unreadable] [unreadable] [unreadable]